The primary objective of this project is to advance understanding of viral evolution in vivo by studying hepatitis C virus (HCV) sequences during the transition from acute to chronic infection, and examining the underlying mechanisms that drive and constrain viral variation. We will measure humoral and cellular host immune responses, as well as the functional impact of immune-driven variation on essential viral enzyme activities. The results will advance our understanding of the host-pathogen relationship, correlates of immune protection, and viral evasion. We hypothesize that HCV evolution is largely deterministic. Specifically, stochastic (discrete and randomly-generated) mutations are filtered by selection, which is mediated by measurable factors that both drive (positive selection) and constrain (negative selection) the resulting variation. Understanding these factors will facilitate rational vaccine design, and guide the use of small-molecule inhibitors of HCV replication. We propose the following aims: (1) to determine the role of the cellular immune response in driving HCV evolution during the transition from acute to chronic infection;(2) to investigate the mechanisms for evolution in the envelope genes of HCV during chronic infection, and the effects of HIV co-infection on HCV evolution, we will study humoral responses to HCV before and after HIV infection, with carefully-matched control subjects;and (3) to investigate the impact of immune escape on viral fitness and the mechanisms of negative selection, we will measure the impact of immune-driven variation on viral replicative fitness, using novel cell culture and single-round subgenomic replicons models of HCV replication. We have already demonstrated that we can obtain the critical tissues required in this investigation, conduct unique longitudinal studies of persons transitioning from acute to established infection, measure cellular and humoral immune responses, and use in vitro models to elucidate HCV pathogenesis. Therefore, we anticipate success in these unique studies of positive and negative selection acting on an infectious agent during human infection.